Amyloid-β aggregates activate peripheral monocytes in mild cognitive impairment

The peripheral immune system is important in neurodegenerative diseases, both in protecting and inflaming the brain, but the underlying mechanisms remain elusive. Alzheimer’s Disease is commonly preceded by a prodromal period. Here, we report the presence of large Aβ aggregates in plasma from patients with mild cognitive impairment (n = 38). The aggregates are associated with low level Alzheimer’s Disease-like brain pathology as observed by 11C-PiB PET and 18F-FTP PET and lowered CD18-rich monocytes. We characterize complement receptor 4 as a strong binder of amyloids and show Aβ aggregates are preferentially phagocytosed and stimulate lysosomal activity through this receptor in stem cell-derived microglia. KIM127 integrin activation in monocytes promotes size selective phagocytosis of Aβ. Hydrodynamic calculations suggest Aβ aggregates associate with vessel walls of the cortical capillaries. In turn, we hypothesize aggregates may provide an adhesion substrate for recruiting CD18-rich monocytes into the cortex. Our results support a role for complement receptor 4 in regulating amyloid homeostasis.

Raw experimental data from NTA analysis are included in Source Data, including technical replicates for all experiments.All single experiment report summary files generated by NanoSight software for analysis of the MCI cohort are included as supplementary files with an anonymous patient number indicated.Video recordings make up more than 300 GB of data and are available upon reasonable request.SPR raw data is included in the Source Data.The gating strategy for flow cytometry and single stains are included in the Supplementary Section, and raw data are provided with Source Data.Flow cytometry fcs files are available upon reasonable request.Dataset obtained from public repositories used in the study included structural data with accession numbers #6SHS and #4NEH from the Protein Data Bank (www.rscb.org).
Gender or sex was not used as criteria for collection of patient or healthy control samples.Collected information stratified according to sex was not analyzed as groups were too small to permit such studies.
Information relating to race, ethnicity, or social groupings were not investigated in our study.
Information on population mean age, sex distribution, and other demographic descriptors are shown in Suppl.Table 1.The APOE genotype of the MCI patients, of relevance for development of Alzheimer's Disease, was analyzed as shown in Suppl.Fig. 6.MCI subjects were recruited from Dementia/Memory clinics in Jutland and Funen, Denmark, and by newspaper advertisements.Subjects were included if they presented with a history of declining memory function over a minimum of 6 months, preferably corroborated by an informant and in the absence of a history of recreational drug use, sedative medication, depression, stroke or systemic diseases.Further inclusion criteria were: (i) age 50-85 years; (ii) %7 years of education or good working history; (iii) meets Petersen criteria (Petersen, 2004) for amnestic MCI (no strict memory score cut-off was used); (iv) an informant was available who had frequent contact with the subject and could accompany the subject to clinic visits or be available to talk on the telephone about the subject's memory and complete the interview for Clinical Dementia Rating (CDR); (v) modified Hachinski Ischaemic Scale score $ 4; (vi) Mini-Mental State Examination (MMSE) score 24-30; (vii) Geriatric Depression Scale (GDS-15) score $ 6; and (viii) an MRI examination that excluded MCI arising from structural causes.Exclusion criteria were: (i) significant neurologic or psychiatric diseases; (ii) history of alcohol and/or recreational drug abuse within 2 years; (iii) contraindications to MRI; (iv) significant reductions in serum B12, red cell folate or thyroid function; and (v) use of medication with known anticholinergic effects (which could impair memory) within the last 3 months or a drug that could impair cognition.
Age-matched healthy control subjects were recruited by newspaper advertisements and screened for neurological diseases.The same inclusion/exclusion criteria as MCI were applied, except that healthy controls had no complaints of memory decline.In principle, especially the recruitment from newspaper advertisement embodies a risk of self-selection, e.g., by including "worried-well" individuals in the patient group.This risk was reduced by admitting all participants to a clinical dementia rating test before inclusion in the study.The validity of the cohort was confirmed by a CDR global score 0.5.Likewise, the HC group was clinically tested and showed the expected characteristics for HC.
The "Central Denmark Region Committees on Health Research Ethics" approved the study Protocol no.1-10-72-191-14). in accordance with the Declaration of Helsinki.All participants signed an informed written consent at enrollment in the study.
The use of donor blood for isolation of human monocytes was ethically approved by the Aarhus University Blood Bank (Protocol no.77).

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April 2023

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Blinding
Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies.Here, indicate whether each material, system or method listed is relevant to your study.If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.No data were excluded.

Materials & experimental systems
All biological experiments were replicated at least once.For experiments using human donor material, the experiments were replicated by analysis of multiple donors, the number of replicate stated in the Figure legends.All attempts to replicate findings were successful as confirmed by relevant statistics were relevant.
No randomization was used: all analyses were made using unbiased measuring techniques ensuring an operator-independent result.
Only one genotype of mice were used in this study and therefore no blinding was necessary.For the in vitro analyses made, all analyses were made using unbiased measuring techniques requiring no blinding.
The axis scales are clearly visible.Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Cell population abundance
All experiments were done with 52 weeks old mice on C57Bl6N background.All test animals lived in standardized conditions where temperature, humidity, and hours of light and darkness are maintained at a constant level all year round.The air is about 22 degrees Celsius; the humidity is about 55 percent; and the light goes on and off automatically, thus imitating a day-night rhythm.
No wild animals were used.
Animals of both sexes were taken, each making up 50% of the group.Other than using data from a group of animals with equal sex distribution, sex was not considered in the study, nor was any data disaggregated for sex collected.This descision reflected no priror expectation that sex would form a basis for differences in the experiments made.
No field-collected samples were used.
All animal experimentation was conducted following approval by local authorities of the Federal State of Berlin (X9007/17).

Not used
Samples were thawed, washed, resuspended in staining buffer, stained with antibodies, and washed again before analysis on the flow cytometer.See Supplementary Materials for further details.
For image stream flow cytometry analysis; IDEAS software package (Amnis) For standard flow cytomtry data; FlowJo, version 10.4.2 for PC (BectonDickinson) Cell sorting of microglia was used to separate cells with a low and high abeta uptake.Based on cells single stained with aggregated Hilyte Abeta 488 (AnaSpec cat.AS-60479-01), two limits were set to have 33% of cells in the low Abeta fraction, 33% of cells in the middle, and 33 % of cells with a high Abeta uptake.As a result, samples with a lower abeta uptake would have a shift towards the lower fraction but never below 10 % in the high abeta uptake faction and never below 2,000 events in each gate.
mouse experiments was determined in line with 3R principles for limiting the amount of laboratory to a minimum while still obtaining a dataset of sufficient size to rule out the presence of large A" aggregates in plasma in line with previously published sample sizes of animal of similar genotype[J Biol Chem.2008 May 23;283(21):14826-34. doi: 10.1074/jbc.M710574200.Epub 2008 Mar 24.] Sample sizes with patients were based on the available MCI-cohort material.In vitro experiment samples sizes were based on experience from previous studies published investigating the role of CD18 integrins[J  Immunol.2020 Mar 1;204(5):1345-1361.doi: 10.4049/jimmunol.1900494.Epub 2020 Jan 22].
20047017, Agilent Microscopy polyclonal anti-human Iba1, cat.no.ab5076.Abcam nature portfolio | reporting summary April 2023Animals and other research organismsPolicy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research, and Sex and Gender in information on the approval of the study protocol must also be provided in the manuscript.